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INTRODUCTION

The Joint Institute for Nuclear Research (JINR) is a unique international scientific center hosting a diversity of nuclear physics facilities, which generate ionizing radiations with different physical characteristics. For many years, it has attracted specialists from many countries to conduct fundamental research not only in physics, but also in biology. Accelerated charged particles are an efficient tool forsolving a lot of urgent problems of modern radiobiology.

Already at the early stages of radiation genetics, its classics N. V. TimofeevRessovsky, D. Lea, K. Zimmer and others pointed out the necessity and potential fruitfulness of using different ionizing radiations for solving fundamental problems ofradiation biology and genetics, including clearing up the mechanisms ofbiological action of ionizing radiations and induced mutation process, andfinding the physical events that trigger mutation formation.





Classics ofquantitative radiobiology repeatedly called for using ionizing radiations ofdifferent quality in genetic research. In this connection, N. V. Timofeev-Ressovsky wrote, The elementary discrete changes intheelementary discrete components ofthe genotype (genes) of all the living organisms studied in this respect are induced by all types of ionizing radiation and form a one-hit doseeffect curve. Experiments with radiations of different dose rates and different hardness allowed quite a precise identification ofthephysical phenomenon that triggers mutations: a one-ionization hit into a certain effective volume. Indeed, the use of isotope alpha-sources and, later, neutron generators inradiobiological experiments allowed beginning the solution of a number oftasks within research on the mechanisms of the lethal action of ionizing radiations oncells of different organisms, biological effectiveness of radiations of different quality, andregularities of the induced mutation process. The emergence of charged particle accelerators first of all, multicharged ion accelerators opened wide opportunities for studying the most topical problems of modern radiation biology. The use of multicharged ion beams generated by JINRs accelerators allowed theInstitutes specialists to solve one of the main issues of radiobiology: the problem ofthe relative biological effectiveness (RBE) of radiations of different quality.

The results of the Laboratorys fundamental research carried out in past years were fruitful for solving numerous practical tasks. The accumulated experimental material is of very high value for high-energy corpuscular radiation therapy (proton and carbon ion beams) of malignant neoplasms; however, further research ontheRBE problem is needed. Accelerator-based radiobiological experiments are important forthe standardization of the radiation exposure of staff working inmixed ionizing radiation fields, which is especially urgent due to the necessity oftaking into account the stochastic effects induced by radiations with different linear energy transfer.

Plans for deep space exploration set new problems for specialists in space radiobiology. In this respect, the use of JINRs basic facilities is considered asa unique opportunity for modeling the biological action of space radiations. It is becoming clear that during manned interplanetary flights heavy charged particles that arepart ofthegalactic cosmic rays will be highly dangerous for the crews. Theenergy range oftheparticles coming from the depth of the Galaxy is extremely wide asitextends toultrahigh energies of the order of magnitude of 1020eV. In the near-term outlook, it does not seem possible to provide protection of the organism from their damaging effect by physical shielding methods. Therefore, accelerator-based modeling ofthebiological action of space types of radiation will help solving these important practical problems of space radiobiology.

The book presents an account of the beginning and development of biological research at JINRs accelerators during several decades.

FIRST RADIOBIOLOGICAL EXPERIMENTS AT JINR

JINRs first radiobiological experiments were performed as early as 1959 at the six-meter proton synchrocyclotron of the Laboratory of Nuclear Problems (LNP). The experiments were performed by scientists of the Laboratory of Radiation Toxicology and clinicians of the Institute of Industrial Hygiene and Occupational Diseases under the supervision of the Head of its Laboratory Prof. E. B. Kurlyandskaya and Director of the Institute Acad. A. A. Letavet. In early1960, a laboratory base oftheInstitute was established at the territory of the LNP;

asmall house was built inthe yard oftheLaboratory of Neutron Physics near now the former physical measurement hall ofthe Laboratory of Nuclear Problems (which is still there). Withthesefacilities, the first staff began to work, who were permanent residents ofDubna with thestatus of LNP-attached staff. The researchers were performing comparative evaluation ofthe effect of proton and gamma irradiation onexperimental animal organisms. Such data were a necessary basis for the development ofmeasures ofreducing the harmful effect of corpuscular radiation onthehuman organism and, ultimately, establishment of the standards for staff working inthemixed fields ofionizing radiation.

In the same years, a number of topical issues emerged in the Soviet Union which were related to the beginning of the space exploration era. An urgent need of thefast solution of these problems stimulated conducting large-scale radiobiological research and determined ultimately the work program of JINRs facilities. The Earth satellites and spacecraft launched at that time found a high level of ionizing radiation in the near-Earth space. It was found that there are different types of radiation inspace; they have complicated charge and energy spectra. During the preparations for the first animal and manned space flights, it was not known how multicomponent radiation of complicated charge and energy spectra, including high-energy protons produced by the Sun and coming from the depths of the Galaxy, would influence living organisms. It became possible to solve this problem in the terrestrial conditions by irradiating biological objects at the six-meter proton accelerator (thefirst one in Dubna), which generated proton beams with an energy of up to660MeV. Thisresearch was aimed at estimating the relative biological effectiveness(RBE) ofhighenergy protons that is, it was necessary to find how effective high-energy protons are in comparison with X- or gamma-radiation as regards their effect on living organisms. In short flights in circumterrestrial space, radiation danger is mainly associated with protons making up the Earth radiation belt or generated by solar chromospheric flares. The maximal contribution to the dose is made byprotons inthe energy range of 100700MeV. Therefore, the most urgent task wasto study the influence of protons of different energies on the human organism and look for ways of protecting the cosmonauts from their adverse influence.

During discussions on the whole set of these issues in the USSR government, aprogram of research and ways of its fulfillment were worked out. InDecember1963, the Institute of Biomedical Problems of the USSR Ministry of Health (IBMP) and a special structure, headed, respectively, by Acad. A. V. Lebedinsky and Prof.Yu. G. Grigoryev, were established in Moscow. In Dubna, a stationary laboratory a branch of the IBMP subdivision was opened at the LNP. This newly opened laboratory was infact a research base where different animals (rats, mice, dogs, and even monkeys), vegetable objects, and cultivated mammalian and human cells were irradiated at the LNP Synchrocyclotron withprotons in the energy range of 25645MeV. A group of IBMP physicists provided theguiding of proton beams of different energies and dosimetry of the irradiation of biological objects based onusing tissue-equivalent absorber units.

At this accelerator, experiments were performed by scientists ofnot onlyIBMP but also of other institutes of the USSR Academy of Sciences (ASUSSR), the Academy of Medical Sciences, and the Ministry of Health. According to the Intercosmos program worked out by the AS USSR, this research was actively participated by scientists of Bulgaria, Czechoslovakia, East Germany, Hungary, Poland, and Romania. In these experiments, reactions were studied of different cellular and tissue systems to acute, fractioned, and chronic proton irradiation. Also, modifying influence wasstudied of different types of physical and chemical agents on radiation effects. Much work was done to evaluate radiation danger in short- and long-term space flights, to set acceptable radiation levels, to develop methods of physical protection from cosmic radiation, etc.

In 1967, the first collection of articles entitled Biological Effect of High-Energy Protons was published under the editorship of Yu. G. Grigoryev, which reviewed the results of research at the Synchrocyclotron of the LNP, JINR. On the basis onthese materials, tens of Candidates and Doctors theses were defended and alot of papers and monographs were published. Analysis of the data on the response of organisms cells and tissue systems showed that the effect of high-energy protons is similar to that of electromagnetic radiation gamma- and X-rays. However, therelative biological effectiveness of protons was observed to grow as their energy decreased to 25MeV and below.

In the long-term space flights, as it turned out, galactic cosmic radiation(GCR) can be the greatest danger. It was found that the GCR consists of almost all elements oftheMendeleev Periodic Table.

The maximal contribution to the integral flux oftheGCR heavy nuclei is made by nuclei of the carbon and iron groups, which are accelerated in the space to huge energies. Though the fluxes of these particles are notintense (beyond the Earths magnetosphere, their total yearly flux is about 105particles/cm2), their damaging effect on the organism can be very strong. In that period, the U-300 heavy ion accelerator of the Laboratory of Nuclear Reactions, JINR, allowed studying the specifics of, and thus modeling, the biological effect of GCR nuclei on living systems. G. N. Flerov actively supported a proposal by two consecutive IBMP Directors Acad.V. V. Parin and, after his death, Acad.O. G. Gazenko and offered the recently launched accelerator for radiobiological experiments.

It should be noted that the arrangement of radiobiological experiments attheU-300 accelerator was rather complicated. Heavy ions could not be accelerated above 10MeV/nucleon. To perform radiobiological experiments, an installation was constructed which allowed transporting accelerated beams of nuclei into the atmosphere. Using this facility, it was possible to carry out the precise dosimetry ofparticles. As the range of accelerated ions in living tissues is not greater than300m, special techniques of preparing biological samples (cell monolayers) for irradiation had to be developed. In experiments on microorganisms, mammalian cells inculture, and cornea tissues of small laboratory animals, heavy ions were observed to have higher biological effectiveness than gamma rays and high-energy protons against many tests, including the criterion of inducing chromosome apparatus damage inmammalian cells.

Besides specialists in space radiobiology, a group of radiobiologists from theAllUnion Oncological Research Center (ORC), the USSR Academy of Sciences, worked at JINR. In 1966, on V. P. Dzhelepovs initiative, work was started at theLNP Synchrocyclotron to create the Soviet Unions first medical proton beam for irradiating oncological patients. A team of physicists involved in this work was headed by LNPs scientist O. V. Savchenko; on the ORC part, the work was headed by Prof. I. I. Ruderman, Head of the Radiation Therapy Department. A proton beam was soon constructed, where the first preclinical radiobiological research had to be performed. For this purpose, a team of the Institute of Industrial Hygiene and Occupational Diseases working permanently in Dubna was invited to work at the ORC. In Dubna, the team studied the biological effect of high-energy protons; it was headed by S. P. Yarmonenko (then Candidate of Biological Sciences).

TheLaboratory of Tumor Radiobiology was established at the ORC; its staff worked in close contact with the team in Dubna.

Radiobiological research at the medical proton beam was started in 1968. In experiments on cell cultures and animals with tumors, the main radiobiological parameters of 180-MeV protons were determined, which soon allowed radiation therapy of patients.

The next stage of research by radiobiologists and oncologists in Dubna wasstudying the biological effect of mesons at a beam of the same LNPs accelerator. Priority data were obtained on the relative biological effectiveness (RBE) and oxygen coefficient of this type of radiation, which was considered promising for the therapy of tumors. Later, the biological effect of superhigh-energy neutrons was studied withthelong view of using them for irradiating large radioresistant tumors.

Radiobiologists of Dubna worked in constant contact with their colleagues in Moscow. As before, research was headed by S. P. Yarmonenko, then already Professor. Joint work was done to prove experimentally the method of hypoxyradiotherapy, which was introduced into the clinical practice of radiation therapy at many oncological institutions of the Soviet Union and abroad.

Later, radiobiological research at JINRs basic facilities was successfully continued by JINRs own biologists, who worked at the Biological Research Sector, LNP (established in 1978).

ESTABLISHMENT OF THE BIOLOGICAL

RESEARCH SECTOR

The Biological Research Sector was established on the initiative of Dr. Phys. and Math. V. I. Danilov, Head of the Synchrotron Department, Laboratory of Nuclear Problems (LNP). Dr. V. I. Danilov was then actively studying the effect of magnetic fields of different characteristics on biological objects. Biologists organized intotheMagnetic Research Group worked to a special order of the Ministry ofMiddle Engineering Industry (the USSR Ministry of Nuclear Engineering). They studied the effect of pulsed and alternating magnetic fields on plants, bacteria, phages, human blood lymphocytes, and neural cells (using a mollusk neuron model). Reactions of vegetable objects to the geomagnetic field screening were another subject of research. In these studies, the Magnetic Screen installation designed and fabricated at JINR was used. It reduced the field 105106 times. Thiswork was performed jointly with scientists of the N.G. Kholodny Institute of Botany, theUkrainian Academy ofSciences (Kiev).

Under the conditions of the geomagnetic field screening, a delay inthegermination of seeds of different plants and in the growth of their germs wasthedominating reaction. A decrease in the pool of proliferative cells and extension of their reproduction cycle duration due to prolongation of some phases (mainly the presynthetic and in some plants postsynthetic ones) were observed. Studies ofthe synthesis dynamics of RNA and proteins dominating in these phases ofthecell cycle revealed a decrease in the functional activity of the genome in all the studied plants during theearly prereplicative period. The obtained results show that thegeomagnetic field is a biologically important factor as it has a certain effect onthe processes oftranscription and translation and on the proliferative processes inaplant cell.

Preliminary research on the effect of a magnetic field alternating in time in asawtooth mode with the maximum of 300 Oe on human peripheral blood lymphocyte chromosomes showed that the magnetic field effectiveness depends strongly on the temperature of cell culturing. In the lymphocytes cultured at temperatures from37to41.5 C, a growth in the number of cells with chromosome aberrations was observed with temperature increasing from 38.5 C, especially under the joint effect of the magnetic field and high temperatures.

On the basis of these results, to coordinate activities in biology and medicine and to develop new research fields, the Biological Research Sector was established at the Synchrocyclotron Department, LNP (JINR Order No. 3388 of 29 November 1977). Prof. V. I. Korogodin was invited to head the Sector; since 1986, the sector washeaded byProf.E. A. Krasavin.

Along with continuing magnetobiological research, the Sector started active research at JINRs basic facilities. The main task was to find out the mechanisms determining the differences in the effectiveness of ionizing radiations of different physical characteristics. For several decades, the problem of the relative biological effectiveness(RBE) of ionizing radiations of different physical characteristics had been one ofthe key problems in radiation biology. Although intense research onthe RBE problem concerning radiations of different linear energy transfer (LET) was performed atmany laboratories around the world, the mechanisms determining these differences were not found out. Numerous mathematical models were proposed toexplain the regularities in the lethal effect of radiations of differentLET oncells ofdifferent origins. However, it turned out to be impossible to explain the ambiguous dependence of RBE on LET within the framework of the developed models.

Themain difficulty blocking clearing up the RBE nature was that RBE is determined ambiguously by both physical factors, which underlie the specifics of energy transfer to matter, and by different biological factors. Although it had long ago been established that RBE has double nature, and, moreover, attempts had been made to separate the physical and biological components in order to derive formulas forcalculating the RBE coefficients, the mechanisms determining the differences in the RBE ofionizing radiations of different types were not found out. It was so because thefollowing important fact was not taken into account: the biological component itself can depend on LET. It led to a mistaken belief that the dependence ofRBE onLET is completely determined by the microscopic distribution of the energy of radiation transferred to the genetic structures responsible for the realization of the radiationinduced effect.

In experiments performed at heavy ion accelerators, it was found that the biological effectiveness of different types of ionizing radiation as regards their lethal action of pro- and eukaryote cells is determined by two factors of different nature: physical characteristics of specific types of radiation and biological properties of cells: their ability to recover from radiation damage (E. A. Krasavin, S. Kozubek, K. G. Amirtaev, P. N. Lobachevsky). The main conclusion made as a result of experimental and theoretical research, on the basis of which the RBE problem was solved, was that DNAs ability to repair depends on LET because the character of lethal lesions also changes and depends on LET.

In the late 1970s, it was discovered in the Soviet Union and abroad that the main DNA structure lesions resulting in cell death are double-strand breaks(DSBs). Later, it was also shown that E. coli bacterial cells die at certain conditions as a result of at least one DNA DSB in a chromosome. Taking into account the revealed facts, it was possible to substantiate experimentally the proposition that the lethal damage character changes with changing LET. It was facilitated bythecircumstance that various repair-deficient mutants of E. coli cells had been obtained by that time. Their use allowed studying the influence of different stages oftherepair process on the specifics of the lethal effect of radiation in a wide LETrange.

On the basis of the known regularities in the induction and repair of primary lesions in the DNA of E. coli bacteria under radiation, a mathematical model oftheradiation inactivation of bacteria was developed. With the use of microdosimetry methods, it was shown that in E. coli cells the DNA lethal lesions of themain type forming under gamma irradiation DSBs develop in the process of single-strand break (SSB) repair; they are enzymatic DNA DSBs. The yield of theDNA DSBs induced immediately by gamma radiation is much lower. With particleLET increasing, the DNA DSB quality related to an increase in the complex DSB yield changes.

When a heavy particle passes through a DNA strand, the complex DSBs arecharacterized not only by simultaneous breaks in the main valency chain, butalso bylesions inthebases in the places of the breaks and by sugar lesions. ForE. coli cells ofdifferent genotypes, the proposed biophysical model explained how radiosensitivity, survival curves, and different modifying factors depend on LET. Itwas shown that in the case of gamma radiation, radiosensitivity is determined by theefficiency of the cell repair systems; in the case of heavy charged particles, radiosensitivity isdetermined only bythe physical properties of radiation. The character ofthedependence of theradiosensitivity of cells on LET is closely related to the E. coli sensitivity togamma radiation.

Experimental tests confirmed all the corollaries to the model (E. A. Krasavin, S. Kozubek, K. G. Amirtaev). It was found that

the character of the radiosensitivity of E. coli cells is genetically determined and depends on the type and yield of lethal lesions induced by gamma radiation;

the dependence of the radiosensitivity of the cells with a normal repair genotype on LET can be described by a curve either with a local maximum or without it, depending on the conditions determining the yield of DNA DSBs of enzymatic nature;

the dependence of the radiosensitivity of mutants with a DNA slow repair block has a drooping character for all LET values; the RBE coefficients of different types of radiation are no greater than 1;

for a mutant with an efficient repair system, a dependence with a sharp maximum is characteristic. For LET 100 keV/m, the radiosensitivity of all the cell strains levels off independently of the repair genotype due to the induction ofstraight complex DNA DSBs.

The developed model conceptions became a basis for studying the factors determining the shape of the survival (S) curves of E. coli cells depending on thedose(D) of radiations of different LET. Research on the influence of different factors of different nature on the shape of the dependence S(D) in E. coli cells showed that this dependence is exponential when in the process of the repair of each radiationinduced lesion, nonrepaired lesions remain which are described by the Poisson distribution. The slope of the exponential curve describing cell radiosensitivity corresponds in this case to the yield of irreparable DNA damage. The limits of cell sensitivity togamma radiation are determined by the yield of the direct DSBs and thesize ofthesensitive target. The survival curves of E. coli bacteria are nonlinear on a semilogarithmic scale, which can be caused by a number of biological mechanisms realized at the population, cell, and molecular levels. The most significant reasons determining the sigmoid character of the S(D) dependence are the specifics ofthebacterial chromosome replication that lead to genome amplification. Along with the amplification factor, the recombination type of repair on homologous parts of DNA can participate in the formation of the arm of the survival curve of cells with a normal repair genotype. With an increase in LET, the survival curves of E.coli bacteria, which are sigmoid in the case of gamma irradiation, undergo characteristic changes: the curve arm diminishes; the slope increases in the intermediate energies and slowly decreases with a further increase in LET. It was found that thetransformation ofsigmoid curves into exponential ones is caused by an increase in thefluctuation ofheavy charged particle energy over the sensitive microvolumes of cells, which leads to the following: as a result of a heavy particle hitting a nucleoid ofabacterial cell containing several copies of the genome, all the sensitive targets are inactivated.

The different role of physical and biological factors in the lethal effect of radiations differing in LET is clearly seen under a number of modifying factors. First ofall, it applies to the modifying influence of oxygen and the influence of two classes of radioprotectors: aminothiols and polyatomic alcohols. Within the framework ofthe proposed biophysical model, an analysis of the realization of the oxygen effect in E. coli bacteria with different repair genotype and the effect of radioprotectors was performed. It was shown that the diversity of the displays of the oxygen effect in cells with different repair genotype is determined in different conditions bytheyield of lesions that are not modified by oxygen and lesions that are not eliminated bypolA-dependent repair. It was also shown that the yield of such lesions grows ascells are exposed to radiation with increasing LET, which results in thesharp attenuation of the oxygen effect when cells are irradiated with heavy charged particles.

Results of research on the action of radioprotectors (cysteamine and glycerin) show that their protective effect is determined genetically: most of the mutations leading to a greater radiosensitivity of cells eliminate or sharply diminish the protective effect of amiothiols. This is because their radioprotective effect is realized at the level of enzymatic repair processes, not at the level of physical and chemical processes. The radioprotective effect of glycerin is also genetically determined, but, unlike the case of cysteamine, this determinancy does not consist in the diminishing or disappearance of the effect of radioprotectors; rather, it consists in its enhancement inthe following E. coli strain sequence: rec A-mutant wild type polA-mutant. Asimilar trend was also found for the oxygen effect. Investigations of repair mutants confirmed the understanding that the protective effect of glycerin is a result of physical and chemical processes which is determined by the protectors ability to block OH radicals and decrease the yield of damage eliminated by the pol A-dependent repair. The established differences between the reactions ofthe strain triad recA-mutant wild type polA-mutant to protectors functioning at the level of physical and chemical reactions and protectors whose effect is based on enzymatic repair processes allowed proposing a scheme of experiments tofind out the mechanism oftheeffect of radioprotectors of different classes.

The main conclusion which was made based on studies of E. coli cells was that the RBE of ionizing radiations of different LET is determined not only by physical characteristics of radiation, but also by biological properties of cells that is, their ability to repair radiation-induced damage. This ability depends on LET, asthecharacter oflethal damage also changes depending on LET.

Since this conclusion had been based on the research performed on prokaryote cells, it seemed important to find out if it was true for eukaryote cells. Experiments onisogeneic strains of haploid yeasts wild-type cells and the rad6 radiosensitive mutant showed that the dependence of cell radiosensitivity on LET, like inthe case ofprokaryotes, is determined by the genotype of cells (P. N. Lobachevky).

Contrary toawild strain, for which this dependence has a local maximum, the rad6 mutant was for the first time shown to have a drooping dependence. The RBE coefficients ofradiation differing in LET are not greater than1 for this mutant. Ashaploid yeasts donot have DNA DSBs repaired in the stationary phase, and the formation ofoneDSB in the genome of such cells results in a lethal event, the higher sensitivity ofthe rad 6 haploid mutants to gamma radiation is caused by disorder in specific stages ofDNA SSB repair. It was shown that this fact can lead to an increase intheyield oflethal DNA DSBs in such cells. Therefore, the dependence ofcell radiosensitivity onLET with a local maximum is observed to transform into adrooping curve.

Onthe basis of the performed research, a conclusion was made that themechanisms determining the difference in the biological effectiveness of radiation for bacteria and haploid yeasts are much alike.

The important role of restoration processes in the biological effectiveness ofradiations of different quality in mammalian cells was revealed in investigations ofthe radiosensitivity of Chinese hamster cells, which were irradiated with heavy charged particles of a wide LET range in the presence of inhibitors of DNA reparative synthesis: cytosine arabinoside (Ara-C) and hydroxyurea (HU) (R. D. Govorun, E. A. Nasonova). The mechanism of the sensitizing effect of Ara-C in aggregate with HU consists in suppressing the reparative synthesis of short gaps in DNA, which results in an increase in the yield of enzymatic DNA DSBs under the gamma irradiation of cells. During the use of radiations in a wide LET range, it was found that under the effect of these agents, the radiosensitivity of cells to gamma radiation increases, while to heavy charged particles, it does not. This causes a decrease intheRBE coefficients of heavy particles when cells are irradiated in the presence of DNA synthesis inhibitors. On the basis of this research, it was shown that, like inthe case of prokaryote and lower eukaryote cells, changes occur in the spectrum of the lethal DNA lesions induced in mammalian cells by radiations of different LET: cluster-type DNA DSBs are formed, the repair of which by cells is either impossible or is extremely obstructed. The analysis of the obtained experimental data based ontheconcepts admitting the induction of only one type of DNA DSBs (directDSBs) by different types of radiation showed that such model concepts do not match experimental results.

The research performed for the first time on lower and higher eukaryotes, aswell as on prokaryotes, showed that the differences in the biological effectiveness of radiations of different LET are determined not only by the physical nature factor thecharacter of the energy deposition by ionizing radiations in genetic structures but also by the ability of the cells to repair DNA damage. Taking it into account, aconclusion was made that the mechanisms determining the RBE nature inproand eukaryote cells are similar in their basic features. But there is no doubt that amore complicated organization of the eukaryote cell genome determines a more complicated radiobiological response of these cells to radiations of different LET.

On the basis of the obtained knowledge of the specifics of the lethal effect of radiations of different quality on cells with different genotype, experiments had been planned to study the mechanisms of the mutagenic effect of radiations in a wide LET range. The mutagenic effect of radiations of different LET was not actually studied in the 1980s. Although it was known that mutagenesis induced by radiations of different physical characteristics is influenced by physical and biological factors, the regularities in the mutation process and the relative role of physical and biological factors in it were not studied. To solve these problems, mathematical models ofthe lethal and mutagenic effect of different radiations on bacteria were developed;

and research was performed on the regularities and mechanisms of the induction of direct and reverse mutations in prokaryote cells (E. A. Krasavin, S. Kozubek,

K. G. Amirtaev, M. N. Bonev, B. Tokarova). The results of these studies are the following:

The dose dependence of the cell mutation frequency under gamma irradiation has a linear quadratic character, which does not change as LET increases.

The relative genetic effectiveness of radiations increases with an increase inLET and is described by a curve with a local maximum, which is shifted to a lowerLET range as compared to a similar dependence for the lethal effects of irradiation.

Mutagenesis induced by radiations of different LET is determined bytheefficiency of the cell repair systems, with inducible SOS repair playing thedecisive role in mutagenesis.

An increase in the genetic effectiveness of radiations with an increase inLET iscaused by a rise in the yield of DNA lesions that are repaired only with theparticipation of the mutagenic branch of SOS repair.

Gene mutations in prokaryotes associated with heavy particle tracks areinduced by the delta-electron region.

The difference in the locations of the maximums of the RBE dependence onLET for the mutagenic and lethal effects of radiation is determined by the different character of DNA lesions. In the former case, they are mainly damaged bases;

inthelatter, DSBs.

The biological effectiveness of radiations of different LET concerning gene mutation induction is determined by the specifics of the microdistribution of energy in the genetic structure, genome condition, and efficiency of the means of repair.

The influence of the biological factor on mutagenesis depends on LET.

The series of research on the mechanisms of the mutagenic effect of ionizing radiations of different physical characteristics on cells with different levels ofthegenome organization won JINRs First Prize in 1987.

DEVELOPMENT OF THE BIOPHYSICS DEPARTMENT

AT THE LABORATORY OF NUCLEAR PROBLEMS

The expansion of the scope of radiobiological research at JINRs basic facilities required a structural reorganization of the subdivision performing this research.

In1988, the Biological Research Sector, at the initiative of its Head Prof.E. A. Krasavin, was restructured into the Biophysics Department of the Laboratory of Nuclear Problems (LNP) (JINR Order No.

248 of 25 March 1988). Atthat time, one ofthepriority research fields of the Biophysics Department was the mutagenic effect of radiations oncells of higher eukaryotes, including humans. It was found that ionizing radiation induces the widest spectrum of mutational variability as compared with other mutagens. It increases the frequency ofchromosome aberrations, as well asgene and genome mutations. It is the mutagenic effect ofradiations on cells ofhigher eukaryotes including humans that is studied the least. Although this effect was discovered in themid-1920s, it is still poorly studied. First ofall, it is so asregards the mutagenic effect of different ionizing radiations with high linear energy transfer(LET) inparticular, accelerated heavy ions.

Induced mutagenesis is a real health and life hazard because not only do newly emerging mutations have an immediate negative effect, they also influence following generations. Mutagens of physical and chemical nature induce a wide spectrum ofhereditary lesions, which, as it is generally acknowledged, underlie the malignant transformation of cells and carcinogenesis, and the development of hereditary diseases in following generations. In both cases, the biological consequences are significantly delayed in time from the immediate action of damaging agents.

The task of studying the mutagenic effect of ionizing radiations, especially on mammalian and human cells, is quite complicated and requires both a wide use of the existing methods and the development of new approaches to its solution. Awide variety of techniques and test systems are now available to geneticists (polymerase chain reaction, preparative column chromatography, blot analysis, fluorescent insitu hybridization technique, and a number of methods of chromosome aberration analysis), which allow studying the mechanisms and the main regularities in radiation mutagenesis in human and mammalian cells.

JINR accelerators offer unique opportunities for conducting research in these fields. At beams of different types of charged particles of wide energy and LET ranges, the Biophysics Department started experiments on cultures of mammalian cells and human peripheral blood lymphocytes. The experiments allowed the main regularities to be found out in the formation of the so-called unstable chromosome aberrations (dicentrics and rings, some types of exchanges between chromosomes), which are detected with a commonly used, classical metaphase method of chromosome analysis. As is known, the quantitative analysis of dicentrics is used for biological dosimetry purposes in cases of accidental uncontrolled irradiations of people.

Thistechnique is also recommended by the World Health Organization to evaluate the state of the environment. But the possibility of estimating the absorbed dose isrestricted in this case by the acute period following an irradiation exposure because the chromosome aberrations lead to disorders in cell division processes andare rapidly eliminated from the irradiated cell population.

It had long been impossible to detect the so-called chromosome aberrations (for example, translocations), which can remain for a long time in a population ofirradiated cells and transfer distorted genetic information from one generation ofcells to another. It is only the fluorescent in situ hybridization (FISH) technique that allowed such stable aberrations to be detected using DNA samples that arespecific forseparate chromosomes of the human genome. Experiments at proton and heavy ion accelerators involving this technique allowed a quantitative analysis ofthefrequency of the formation of the translocations of some chromosomes depending onthe radiation dose and LET (protons, nitrogen ions, and gamma rays).

Itistheformation of stable chromosome aberrations that are considered now toinitiate anumber of oncological diseases, for example, chronic and acute myeloid leukemia. Thequantitative assessment of stable chromosome aberrations in organisms cells can also be considered a reliable method of retrospective biological dosimetry foraccidental uncontrolled irradiations of an organism.

At the Biophysics Department, comparative studies were performed of the regularities in the induction of mutations in the mammalian cell gene HPRT depending on the initiating dose of radiation, including heavy ions, in a wide LET range (R. D. Govorun, P. N. Lobachevsky, N. L. Shmakova). It was found that heavy ions and gamma rays have a high mutagenic effect on mammalian cells. The obtained data on the survival rate of Chinese hamster cells and the frequency of mutations induced by gamma rays and accelerated 4He and 12C ions of different LET showed that the character of the mutation induction dependence on the radiation dose and LET is ambiguous: for gamma radiation, the doseeffect curve has, according to the mutation induction criterion, a pronounced nonlinear (power law) behavior. Thecurves were also observed to be nonlinear for heavy ions with LET values of20and 50keV/m. For higher LET, depending on the dose, the mutation induction curves become linear. The survival rate curves are, respectively, sigmoid and become exponential. These experiments showed that as regards both the cell survival rate and the mutagenic effect (chromosome aberrations and gene mutations), the maximal biological effectiveness corresponds to LET of about 100 keV/m, RBE with respect to the mutagenic effect being twice as high as RBE with respect tothe survival rate.

In that period, a great amount of preclinical radiobiological research wereperformed at the medical beams of the LNP Synchrocyclotron (N. L. Shmakova, T. A. Fadeeva). It required evaluating the biological effect of charged particles onnormal and tumor cells of mammals. It was possible to perform this kind ofresearch onanimals inoculated with tumors. Experimental animals can be inoculated with a great amount of different tumor lines; then it is possible to study their response to irradiation and estimate the damage to normal tissues, for example, marrow, which is the critical radiosensitive system in radiotherapy. It is such experiments that were performed first of all. But experiments in vivo on whole organisms showed that itisimpossible to make a precise quantitative estimation ofthemain parameters of the biological effect of particles: RBE and oxygen enhancement ratio(OER) because the individual radiosensitivity of cells and the organisms general influence on the tumors response had a too strong impact on the experiment results. In this connection, a task arose of studying the main biological parameters of medical beams using cells that had been isolated from an organism and were growing invitro on artificial nutrient media that is, cell cultures. It required construction of special rooms and sterile boxes because a possibility of bacterial contamination ofcell isvery high.

The clonogenic ability of cells and chromosome apparatus damage were taken asthe main quantitative indicators for evaluating the biological effect of radiation.

The RBE of protons and mesons in the Bragg peak and at the beam entrance were compared with the effect of gamma and X-rays that are commonly used inradiotherapy.

A major problem in radiation therapy consists in overcoming the radioresistance of hypoxic cells, which are, as a rule, present in the tumors due to the poor development of the vascular network of the neoplasms. Under sparsely ionizing radiations, gamma and X-rays, and protons, the difference in the radiosensitivity of oxygenated and anoxic cells is notable; OER is then equal to3. Under radiations with high LET, the OER decreases. By that time, no data had been published on the OER for+and

mesons. It should be noted that determining this magnitude, which is very important in radiotherapy, requires complicated technical contrivances. Itis necessary to compare the radiosensitivity of oxygenated and anoxic cells, where the oxygen content should not exceed 2030 parts per million. For this purpose, special equipment was designed that evacuated the gas mixture from vessels containing cell suspensions and then filled them with nitrogen. Although this procedure was performed repeatedly, attempts to achieve an OER value of3under gamma radiation failed. Itis only after the replacement of superpure nitrogen with argon and increasing themetabolic consumption of oxygen from the environment by providing a high cell concentration that the necessary OER value of3 was achieved for gamma radiation, and the OER was found to be 1.7 for mesons. It was the worlds first research ofthis kind; later, the data were confirmed by American scientists.

In parallel with these studies, intensive work was being done to master theuse ofartificial hyperglycemia(HG) to increase the efficiency of radiation therapy oftumors (V. I. Korogodin, N. L. Shmakova, T. A. Fadeeva). It is known that the metabolism ofthe tumor cells, contrary to that of the normal cells, exhibits an increased glycolysis. As the glycolysis product, the lactic acid is produced; its accumulation results in tumors self-acidulation. The research on animals inoculated with tumors performed by von Ardenne (Germany) and scientists of the Byelorussian Oncological Institute showed that, if the sugar content in blood is artificially raised, the radiation therapy efficiency significantly increases. This dependence was generally believed to be associated with the suppression of postirradiation repair in decreased pH.

However, noexperimental facilities adequate to the clinical application ofthemethod and stable reproduction of results were available. The aim of the Biophysics Departments experimental research was to study the cell mechanisms ofHG. Theexperiments were performed invitro on tumor cells taken from animals immediately beforeanexperiment. Byapplying a graduated glucose load and changing theoxygenation level, itwas possible tocontrol the level of acidulation and survival rate of the tumor cells. The experiments yielded remarkable results. It was shown that as the degree ofacidulation observed in hypoxia is achieved, tumor cells die onamass scale without any external influence, in particular, irradiation. Quantitative estimations showed that the additivity of two effects exists: hypoxic cells die due to selfacidulation; well-oxygenated cells, which undergo glycolysis slowly, die as a result of irradiation. NoHG influence on postirradiation was observed. These results were confirmed by a number ofspecialized oncological laboratories.

The use of vasoconstrictors, which additionally increase hypoxia in tumors and enhance glycolysis, doubled the efficiency of therapy. Unfortunately, this research was not continued, because due to the circumstances of the early 1990s the teams concerned with this subject broke up.

Along with radiobiological research on cells of higher organisms, the Biophysics Department performed comprehensive work on cells of lower eukaryotes: yeast cells (V. I. Korogodin, N. A. Koltovaya, V. L. Ilyina). Yeast, which is a type of fungus, isone of the most common objects of research on living organisms. Man has always faced adverse effects of different pathogenic organisms, but yeast seems to have been thefirst microorganism to be used for mans practical needs.

It is well known that ploidy is one of the main factors determining the specifics ofyeast cell response to irradiation. The diploid cells differ from the haploid ones in radiosensitivity, the survival curve shape, RBE, and manifestations of the mutations increasing the cell sensitivity to ionizing radiations. Attempts to interpret theseregularities led to a suggestion that diploid cells have the so-called diploid-specific damage repair. This problem has an important general biological aspect. During evolution, the transition from haploid organisms to diploid ones led to asignificant increase in the stability of the genetic apparatus against different external damagingfactors.

At the Biophysics Department, research was performed on the role of the factors determined by the diploid condition of the genome in the sensitivity of yeast cells withdifferent genotypes to ionizing radiations of different LET. It was shown that the regularities in the lethal effect of ionizing radiations of different quality on diploid yeast cells are caused by at least two diploid-specific processes: damage repair underlying the postirradiation restoration of cells and the processes determining theaftergrowth effect. The research allowed a conclusion to be made thattheprocesses of radiation damage induction and repair are mutually independent. Forthefirst time, an evaluation was made of the role of diploid-specific repair intheradiosensitivity of cells under high-LET radiations, and the dependence oftheefficiency of repair processes on the radiation quality was studied. It wasshown that under high-LET radiations the sigmoid form of the survival rate curve ofdiploid cells is determined exclusively by the aftergrowth effect.

Later, research on the mutagenic effect of ionizing radiation in yeast cells wascontinued (N. A. Koltovaya). The problem was that it is rather difficult to establish thenature of a mutation-type lesion. During this work, genetic systems weredeveloped that allowed the exact determination of the nature of a mutation event without using expensive and labor-intensive techniques.

As a model system for studying total mutagenesis, the CAN1 gene was used.

Itcodes arginine-permiase and is 1800 base pair-long. Mutations of any nature lead to a disorder in the functioning of this gene and development of antibiotic resistance.

For testing large restructurings, two test systems were used that allowed detecting mitotic crossingover and conversion. As is known, mitotic recombination isinduced by double-strand breaks (DSBs). Recombination results in the formation ofextended changes in genetic material.

The analysis of microdeletions was based on using reversions in strains thathave frameshift mutations. The used strain carries an insertion of the fourth base intotheLYS2 gene and + 1T insertion into the 6T sequence in the HOM3 gene.

To evaluate the induction of the nucleotide substitution type of point mutations, a test system developed by Prof.M. Hampsey (Louisiana State University) was used.

The system is based on the position of cysteine22 in the cytochrome-c protein being critical. Six strains were constructed with base substitutions in this position, which led to enzyme inactivation and impossibility of growth on a medium with an unfermentable source of carbon. The functional activity can be restored only by true reversions restoring the cysteine codon in position22. Reversions in the CYC1 gene of each of the six strains represent one of the six possible substitutions of base pairs.

Thus, a simple and reliable system was created which allows identifying changes inthe DNA nucleotide sequence without using complicated molecular and genetic techniques.

In experiments on this system, the spectrum of mutations induced by gamma irradiation was studied in most detail. Most efficiently, ionizing radiation induces large restructurings, whose frequency is of the order of 1%. Among the nonextensive mutation events, it is, of course, mutations in the CAN1 gene that are induced most efficiently, which reflects the sum character of the mutations. A linear dependence of forward mutation yield in the CAN1 gene on the gamma radiation dose was observed. An analysis of the mutation spectrum showed that compared withspontaneous mutations, the gamma-induced mutation spectrum has an increased proportion of the ATTA transversions. The spectrum of the base pair substitutions inhaploid and diploid yeast strains is the same. The maximal contribution (more than30%) is provided by the GCAT transitions. The induced mutation spectrum does not depend on the dose.

Another field of research concerned the repair mechanisms of radiation-induced DNA double-strand breaks (DSBs). It was found that yeast cells have not only a slow type of DSB repair, but also a fast one. It was shown that both slow and fast types ofDSB repair are efficiently realized only in diploid yeast cells.

A third field of research was focused on the regularities in spontaneous mutagenesis (V. I. Korogodin, A. I. Chepurnoy, V. L. Korogodina). For research, genes were chosen that control the synthesis of adenine and leucine. The initial strains areauxotrophic and are unable to grow on a medium without an addition of thecorresponding product. Reversions to prototrophicity can be realized in two ways: bytheformation of reverse mutations in the gene controlling its synthesis and by forward mutations in suppressor genes. It was found in special experiments that, when theactivity of the gene is suppressed, the frequencies of this gene forming mutations are two orders of magnitude lower than when it functions actively. At the same time, suppressor genes, whose activity does not depend on the adenine presence in the medium, mutate with approximately the same frequencies in both cases.

Thus, the Biophysics Department performed versatile radiobiological research at JINRs basic facilities. After it had become possible to accelerate heavy nuclei torelativistic energies at the Phasotron of the Laboratory of High Energies and physical experiments had been started there, radiobiological experiments were proposed for high-energy heavy ion beams. This required special spectrometry and dosimetry studies of relativistic heavy nuclear beams. The staff of the JINR Department of Radiation Protection and Radiation Research already had great experience inthis field. The JINR Directorate V. G. Kadyshevsky and A. N. Sissakian (JINR Director and Vice-Director of the time, respectively) supported an idea to merge theLNP Biophysics Department and JINR Department of Radiation Protection and Radiation Research into JINRs new subdivision: the Department of Radiation and Radiobiological Research (JINR Order No. 270 of 27 April 1995).

JINRS DEPARTMENT OF RADIATION AND

RADIOBIOLOGICAL RESEARCH

The main tasks of the Department of Radiation and Radiobiological Research (DRRR) were the following:

continuation of research on the regularities and mechanisms of the genetic effect of ionizing radiations with different physical characteristics;

research on the interaction of radiation with matter and development ofradiation monitoring methods;

monitoring of radiation environment at JINRs subdivisions to provide radiation safety according to the standards and regulations on the use of radioactive materials and other sources of ionizing radiations in force in the country of JINRs location;

development of radiation monitoring systems for JINRs new and reconstructed (upgraded) nuclear research and radiation-dangerous facilities and sites.

To realize its main functions, the DRRR, in particular, designed equipment forradiation and radiobiological research, performed experimental data processing, and carried out theoretical research to model the interaction of radiation with matter.

Radiobiological research In radiobiology, research on the mutagenic effect of radiations in a wide linear energy transfer (LET) range was continued. In experiments on bacterial cells, regularities in structure (deletion) mutations and mechanisms of their induction were studied (A.V. Boreyko). It is quite a topical issue because to standardize theloads ofradiations of different quality on staff working in mixed ionizing radiation fields, to provide radiation safety of cosmonauts on long-term missions, and to solve anumber of other important practical problems is very important not only tohave information on the total yield of different kinds of mutations inirradiated cells; also, comparative data on the frequency of gene and structure mutations areextremely interesting. Studying the dose dependences of the point and chromosome mutation yield in higher eukaryote cells under ionizing radiations inawide LET range isarather difficult and laborious task requiring the use of complicated molecular biology methods. Obtaining this kind of information ismuch easier inexperiments onprokaryote cells. Using accelerated heavy ions, it was shown that deletion mutation formation frequency increases linearly withthe dose for all types ofradiation;

deletion mutations are induced most efficiently by ions witha LET of6080keV/m.

It pointed to the different character of the DNA lesions underlying the induction of gene and deletion mutations. The former are cluster lesions of a DNA strand;

thelatter, DNAdouble-strand breaks (DSBs).

In experiments on yeast cells, adaptive and induced mutagenesis mechanisms were studied (V. I. Korogodin, N. A. Koltovaya, V. L. Korogodina, A. I. Chepurnoy).

During several years, lively debates about the nature of adaptive mutations inmicroorganisms took place in literature. At first, the adaptive (directed) mutations were determined as mutations emerging only in the presence of selective pressure orinslowly growing cells in the stationary phase. But it was shown on E. coli bacterial cells that nonselective mutations can also emerge at an unexpectedly high rate. It was shown later that starvation increases the frequency of both selective and nonselective markers. According to the concepts developed at the DRRR, the socalled adaptive mutations are not adaptive; rather, they emerge due to the transitional hypermutable state of cells under stress: the advantaged mutations are immediately selected; other mutants die quickly. In cooperation with the specialists oftheUniversity ofPerugia (Italy), research was done on the genetic control of mutagenesis under starvation, to which yeast cells respond by stopping division and entering thestationary growth phase. This research was closely related to studying the genetic control ofthecell cycle arrest following DNA damage. In recent years, a connection becomes more obvious between different components of the integral cell response to DNA lesions, which provides genome stability and integrity. The relation was shown between thecell cycle control mechanisms and the DNA damage repair mechanism. Thisrelation checkpoint control allows cells to survive and maintain genetic stability; it is regulated by checkpoint genes. It is believed that a disorder in checkpoint ways that leads to an increase in mutability and genome instability is very important attheearly stages of carcinogenesis.

Research performed by different laboratories resulted in the understanding of the molecular mechanisms of checkpoint control responding to DNA damage. It is assumed that the RAD9, RAD17, and RAD24 genes participate in the early stages of DNA lesion detection. The RFC-Rad24 protein complex seems to realize the loading of the Rad17-Mec3-Dbc1 protein complex or repair enzymes intotheplace ofaDNA lesion. The kinases in particular, RAD53 participate in signal transmission; the CDC28 kinase functions at the final stages of the cell cycle arrest regulation, which is necessary to repair the damage. It is assumed that adisorder in cell cycle arrest leads to genetic instability and an increase in cell sensitivity to the damaging agents. The latter was observed in most of the mutants with a deficient regulation of cell cycle arrest. This increase in cell sensitivity to the damaging agents, though, could be related not only with the absence of cell cycle arrest after a lesion, but also with the participation of some checkpoint genes in repair.

Thebranched scheme of the genetic control of cell cycle progression and arrest needs intensive research. Jointly with the Institute of Molecular Genetics (the Russian Academy ofSciences), the DRRR studies thegenetic control of the checkpoint ways and theirinfluence oncell sensitivity tothedamaging effect of radiation. An analysis oftheinteraction between the RAD9, RAD17, RAD24, RAD53, and CDC28 genes showed that the RAD9, RAD17, and RAD24 genes belong to the same branch which determines cell sensitivity to gamma radiation, although the RAD9 and RAD24 genes belong to different branches which determine cell sensitivity to UV rays and MMS and cell cycle arrest. TheRAD53 and CDC28 proteinkinases are epistatic relative totheRAD9 gene, but they are more likely to belong to different branches determining radiosensitivity. According to literature data, the CDC28 and RAD53 genes belong to the same branch determining cell cycle arrest. The data obtained point tothe divergence oftheways of the regulation of cell cycle arrest and radiosensitivity. These data show that the genes are multifunctional, and their participation intheintegral response does not add up to cell cycle arrest; some of them also take part inrepair processes.

After DRRR establishment, large-scale cytogenetic research was started on mammalian and human cells (R. D. Govorun, N. L. Shmakova, I. V. Koshlan, M. V. Repin, N. V. Koshlan, T. A. Fadeeva). As was already mentioned, the mutagenic effect of high-LET radiations on higher eukaryote cells had been studied rather poorly.

Themain tasks in this field formulated by the DRRRs specialists were further research ontheregularities in the induction of mutations in the HPRT gene of mammalian cells by accelerated heavy ions, studying the cytogenetic characteristics oftheHPRT-mutant subclones grown from single cells keeping in the following generations the HPRT mutations that developed in them, investigation of unstable and stable chromosome aberrations in human lymphocytes induced by heavy charged particles, and research on the cytogenetic effects of low doses of radiation.

Irradiation of cells with heavy ions and gamma rays revealed the high mutagenic effect of these radiations on mammalian cells. The relative biological effectiveness (RBE) of the studied heavy ions against the effect of gamma rays isdescribed byacurve with a local maximum at a LET of about 80100keV/m. An interesting observation was made in a LET range near 20keV/m for different radiations, wherethe mutation yield curve shifted to higher RBE against tests of cell inactivation and chromosome aberration formation. It had been shown earlier inresearch onbacteria that themutagenic effect maximum of the RBE dependence on theLET of different radiations was significantly shifted to lower LET and corresponded toabout 20keV/m, while the maximal RBE values for heavy ions asregards thelethal effect were observed at 8090keV/m. This fact was determined bythedifferent

character of the molecular lesions underlying the induced mutations and lethals:

in bacteria, the vast majority of the mutations are gene mutations and are mainly related to the lesions of bases, while the lethal effect is determined bythe induction ofDNADSBs. In research on mammalian cells, asimilar character oftheRBE dependences ontheLET of different radiations was obtained against thetests oftheinduction of mutations, chromosome aberrations, and cell inactivation, which can point tothe fact that the same events DNA DSBs underlie the lesions leading tothesame effects inmammalian cells. A shift ofthecurve of the RBE dependence onLET ofdifferent radiations in a LET range near 20keV/m against the induction ofmutations in mammalian cells, as compared with the lethal effect, is determined by an increase in the contribution by the point mutations; at higher LET, gene mutations prevail that are related to different types ofdivision. A similar character of the curves of the RBE dependences on LET against these three tests can point tothesame lesions DNA DSBs underlying these effects in mammalian cells.

They induce chromosome aberrations and mutations like macro- and microdeletion of genes in DNA.

Under the assumption that the mutation process in cells can be accompanied with a disorder in the structure integrity of the chromosome apparatus and show up as the chromosome instability of cells, research had been performed to isolate single mutant colonies, from which subclones were grown. Then, a cytogenetic analysis ofthe subclones was made (R. D. Govorun, I. V. Koshlan), which revealed the heterogeneity of the spontaneous and radiation-induced HPRT-mutant subclones concerning the studied cytogenetic indicators (mitotic activity, aneuploidy, and chromosome aberration level). It was found that the consequences of the mutation events appeared as the development of the genome (against the number of chromosomes in cells) and chromosome (against the chromosome aberration level) instabilities inpopulations of the progeny of mutant cells.

In the detection and selection of mutant subclones, mutants with retarded growth, as compared with the intact control, were observed. Retardation ofthegrowth of many mutant subclones in a selective medium could have been determined bytheformation of gene mutations leading to a decrease in enzyme activity or synthesis ofalower amount of the native enzyme. In such cases, the viability ofa mutant population could have been provided only at the expense of the cells which could not have utilized the purine analog during the cell cycle.

As the criteria of evaluating the mutant subclones with respect to the number of chromosomes in cells, the modal number of chromosomes and the percentage of cells in the corresponding mode were chosen. An analysis of the chromosome spectra revealed marked aneuploidy up to full ploidy. Among the subclones, samples prevailed with the modal number of 21 or 22 chromosomes. The share of diploid mutants with the modal number of chromosomes was70% and more up to100%.

The share of cells in this mode varied largely between the samples. Spontaneous mutants made up5080%, which actually did not differ from the reference value.

Theradiation-induced mutants were especially heterogeneous as regards the chromosome spectra.

To explain the phenomenon of chromosome instability, R. D. Govorun and I. V. Koshlan proposed a metabolic hypothesis. As is known, the cell has two ways of the synthesis of purine nucleotides: the de novo synthesis (a phased synthesis based on ribose-5'-phosphate) and the synthesis from finished products. Forthecell, the latter is more advantageous concerning energy. It is realized with the participation of the native HPRT enzyme. When mutations take place in the hprt locus and are accompanied with the termination of the enzyme synthesis, the production ofpurine nucleotides has to be realized de novo. When the enzyme with decreased activity, oran insufficient amount of the enzyme is synthesized, conditions for thecompetition of the two ways are formed in the cell. A situation develops leading to the violation of the cell metabolism equilibrium. The cell activates the mechanism ofDNA synthesis involving the hprt enzyme, but this enzyme is not functional enough and fails to produce the necessary nucleotides. It leads to metabolic imbalance, whichisa signal for the activation of the equilibrium search mechanisms.

Thedeficiency ofpurine bases in the build-up of DNA chains results intheactivation of synthesis denovo.

This labile state might be accompanied withchromosome instability asastage of the search for equilibrium and adaptation to changed existence conditions. Finally, mutant subclones will be formed with an increased yield ofchromosome aberrations against the reference value. Hence, for the survival ofmutant cells, the full termination of the enzyme synthesis is more favorable (inthecase of a full or a major deletion of a gene) than the synthesis of the enzyme with decreased activity. However, the formation and survival of mutant cells in theorganism leads tothe development ofpathologies. Structural chromosome anomalies affecting specific genes attract special attention as the objects of research because their role in thepathogenesis of tumors in humans in particular, in thedevelopment ofleukemia isbecoming increasingly obvious. From the point of view ofthe metabolic hypothesis, thechromosome instability mechanism in the progeny of mutant cells is getting clearer. Mutations are passed on from one generation to another; thus, the issue of the conservation of the triggering event in the following generations ofmutant cells is resolved.

Much work was done on studying the regularities in the induction of unstable and stable chromosome aberrations in human cells by different types of radiation (R. D. Govorun, M. V. Repin). The unstable aberrations include different types ofchromatid and chromosome exchanges leading to the formation of changed chromosomes that are atypical to the cell population: dicentrics, polycentrics, rings, and different fragments of chromosomes. Their appearance goes along with disorders incell division processes and, as a rule, fast cell death. Unstable chromosome aberrations are analyzed with the standard metaphase method, which allows their detection over the whole genome of the cell during microcopying lymphocytes withausual light microscope. A linear dependence of the frequency of the formation of cells with chromosome aberrations on the irradiation dose was observed forall the studied types of radiation. For the total number of chromosome aberrations, a power dependence of the effect on the dose of sparsely ionizing radiations (protons and gamma rays) was established. Under heavy ions, it modifies into a linear one.

However, under high doses of such radiations, the effects weaken due toasignificant delay in mitoses, especially of the heavily damaged cells with numerous chromosome aberrations.

The stable chromosome aberrations form as a result of a symmetric exchange of sections between two damaged chromosomes that does not lead to a disorder inthe behavior of chromosomes during cell division. In the following cell divisions, such chromosomes behave like normal ones and, with distorted genetic information, are passed on to the following generations of cells. Stable chromosome aberrations, like translocations and insertions (inserting into a chromosome a section of another chromosome), are long kept in cell generations to follow. It is generally recognized that such chromosome restructurings later can lead to the development of mutagenic processes and carcinogenesis in the human organism. In the early20th century, thedevelopment of the fluorescent in situ hybridization (FISH) technique allowed detecting stable chromosome aberrations. Stable aberrations of separate chromosomes stained with fluorescent dyes are detected with luminescent microscopes inthe cell genome. For this purpose, the chromosomes are stained with dyes using specific samples with unique DNA sequences.

In research performed at the DRRR, DNA samples were used that are specific to chromosomes 1 and 2 of the human lymphocyte genome its largest chromosomes. Their damage is more probable under ionizing radiations. With the FISH technique, a high frequency of the formation of translocations (stable aberrations) in these chromosomes was observed. The RBE coefficients of radiations with a LET of80keV/m were 3 and more.

Extensive research was performed on mammalian cells to evaluate the cytogenetic effects of low doses of radiation (N. L. Shmakova, T. A. Fadeeva). As is known, the evaluation of the biological effect of low doses of ionizing radiation is necessary topredict the genetic and carcinogenic risk of irradiation. The difficulties ofestimating theeffects and determining the doseeffect curve of low doses are related totheproblem of the acquisition of the reliable statistics of mild damage induced bysuch doses.

Therefore, the risk associated with low doses is estimated based ontheextrapolation of high dose effects to low doses; the results thus depend onthemodel underlying anextrapolation. The nonthreshold linear concept, which is the most conservative as it suggests that any even the smallest excess ofthenatural radiation background presents danger, is officially accepted and isthebasis ofthe recommendations by the International Commission on Radiological Protection. Butexperimental data of recent years explicitly contradict this concept and show that itisincorrect to extrapolate the effects linearly from high doses tolow ones. Inestimating thebiological effects of low doses, it is the frequency of cytogenetic lesions namely, chromosome aberrations(CA) and micronuclei(MN) incells ofdifferent types that, asa rule, is registered. It is characterized by a clear quantitative dependence in a wide dose range. The dose curves can be reproduced well ondifferent objects; they have aunique feature that consists in the presence ofadose-independent part in the dose range of 0.10.5Gy.

In experiments on peripheral human blood lymphocytes, asynchronous and synchronized populations of V-79 Chinese hamster cells and BRO human melanoma cells, it was shown that the dependences of the quantity of cells with CA on the radiation dose have a similar pronounced nonlinear character. Under irradiation with a dose of 00.05 Gy (lymphocytes), 00.1 Gy (melanoma cells), and00.2Gy (Chinese hamster cells), the chromosome lesion yield sharply increases against thereference level (the hypersensitivity (HS) range); then itnotably decreases and passes intothedose-independent range. Above 0.5Gy, cell resistance increases (whichis theinduced resistance (IR)), and the dose dependence becomes linear. In going from the HS range to the IR range, the slope of the curves decreases 23times for Chinese hamster cells and melanoma and 510times for human lymphocytes depending onthe method used for analyzing CA. Similar doseeffect curves were obtained as other donors lymphocytes were irradiated with X-rays.

Research onthefrequency of different types of gamma-induced aberrations in human lymphocytes showed that HS is determined mainly by an increase intheyield of chromatid aberrations, which prevail below 0.5Gy.

A study of the nature of the HS and IR phenomena performed on Chinese hamster and human melanoma cells allowed a conclusion to be made that the shape of the doseeffect curve of an asynchronous population of Chinese hamster cells concerning CA induction is reproduced well on synchronized cells irradiated intheG1 phase of the cell cycle. It points to HS being determined by the high radiosensitivity ofthe population as a whole in a narrow range of low doses and notbeing related to the death of the fraction of cells that were irradiated in the radiosensitive phase ofthe cell cycle. As the irradiation dose increases, all cells become more radioresistant, probably, due to the induction of repair processes. Thus, the most plausible explanation of the nonlinearity of the doseeffect curve and the transition from HS to IR is that at a certain level of cell damage inducible repair systems are triggered. Itresults in a decrease in the radiosensitivity of cells and slope ofthecurves. Comparing dose dependences of CA induction in Chinese hamster and human melanoma cells provides the grounds to suggest that the inducible systems ofmelanoma cell repair are triggered at lower doses and work more efficiently thanin Chinese hamster cells.

Jointly with radiochemists of the Laboratory of Nuclear Problems V. A. Khalkin and Yu. V. Norseev, an extended research was performed on the biological effect ofastatine-211 and possibility of its use in targeted cancer therapy (N. L. Shmakova, P. V. Kutsalo, T. A. Fadeeva). In the earliest experiments, it was shown that ascitic forms of cancer can be extracted with astatine-211 adsorbed by tellurium.

These results inspired a search for methods of the targeted action of alpha emitters onthemelanoma one of the most aggressive forms of malignant neoplasms, which is characterized by early and vast metastases. For treating metastases, thetargeted use ofastatine-211 is the most efficient technique as its decay produces alpha particles with a range of 60m, which makes up several cell diameters.

Asameans of transporting the radionuclide to the tumor cells, a polycyclic compound, whichisknown in medicine as methylene blue (MB), was used as it has a high capacity of binding the tumor cell melanin. It was shown in vitro using human melanoma cells and normal nonpigmented cells that an astatine-211MB compound was accumulating selectively in melanin-containing tumor cells, which resulted in1520-fold higher damage of melanoma cells as compared to that of healthy cells.

An MB-based iodine-131MB preparation was obtained, which proved tobehighly efficient in visualizing a melanoma and its metastases in animals withan inoculated melanoma. This research was continued for the purpose of introducing thisdiagnostic preparation in clinical practice and the development of methods ofusing the astatineMS compound to prevent melanoma metastases.

Later, two separate sectors were established at the DRRR: the Photobiology Sector (headed by M. A. Ostrovsky) and Computer Molecular Modeling Sector (headed byKh. T. Kholmurodov). At the Photobiology Sector, research was started underthedirection of Acad.M. A. Ostrovsky (Russian Academy of Sciences) onmolecular photo and radiobiological processes in eye structures (the lens and retina).

Taking up such tasks is a new step in the progress of JINRs biophysical studies.

Theurgency of this research is first of all related to the necessity of solving theproblems of space radiobiology. It is increasingly evident that in a long-term space flight, there isahigh risk of cataract development. In this connection, studying the effect ofheavy particles on the aggregation of the lens proteins (crystallins) andthe mechanisms ofsuch an aggregation is a topical problem. Research was started onthe damaging effect of heavy particles on the visual pigment rhodopsin andthefunctional state of the retina.

In connection with the emergence of high-performance computers (supercomputers and specialized clusters, for example, the MDGRAPE-2 system) and multipurpose software packages (DL_POLY, AMBER, CHARMM), it became possible touse computer molecular dynamics (MD) modeling methods in physicochemical and biological systems. One of the important applications of MD methods iscalculating conformational changes in proteins and determining the spatial structureofproteins with high precision. The MD methods allow modeling mutational changes in biological structures at the molecular level with high spatial and temporal resolution.

The MD Sector conducts theoretical research to model the protein surroundings of different isomers of retinal. This research is focused, in particular, on the chromophore group within the retinal-containing proteins first of all, the11-cis retinal.

Radiation research Before the establishment of DRRR, JINRs radiation research was carried out at the Radiation Protection Department (RPD; since 1975, the Department of Radiation Protection and Radiation Research). The 1950s60s was the time of the rapid development of particle accelerators as the most important nuclear physics instruments. The energies of the accelerated particles and the currents oftheextracted beams were continuously growing. Since its establishment, JINR has been developing mainly as a major accelerator center. The IBR-30 reactor did not change this situation in the main, nor later did the second-generation reactor IBR-2 becauseJINRs basic facilities include accelerators of different types offering a wide mass range and a severalMeV10GeV energy range of accelerated particles.

As a scientific discipline and as an activity, dosimetry was formed, first of all, toprovide the radiation safety of the nuclear industry personnel. On a national scale, thestaff exposed to accelerator radiation fields made up a tiny fraction oftheoccupationally irradiated people. Moreover, the complexity and diversity ofaccelerator radiation fields and the necessity of the development of specific means of measuring their characteristics resulted in accelerator-related protection physics and dosimetry growing into a separate area of physics. As regards opportunities forconducting this kind of research, JINR has always been a unique center. For this reason, since the establishment of JINRs RPD in 1963 (the Department was first headed byM. M. Komochkov), most of its activity has been related to accelerator protection physics. This specificity has determined the character of RPDs scientific and practical work for a long time. The formation of this field of science was initiated inthe1950s in connection with the start-up of medium-energy accelerators (the Cosmotron in Brookhaven, Bevatron in Berkeley, and Synchrocyclotron in Dubna). The first experimental studies of the protecting properties of materials, high-energy radiation attenuation by shielding, and other related research were performed at that time. Notheoretical approaches were then available to calculate reliably radiation transport through massive shielding. To predict the accelerator radiation environment, empirical and phenomenological methods of shielding calculation were used.

Experimental data on the development of the internuclear cascade in the shielding volume were very scarce, which stimulated accelerator-based protection physics experiments. At Berkeley and JINR, and later at CERN and the Institute of High Energy Physics (Protvino), a great amount of studies were done mainly toobtain and refine the empirical constants for doing calculations in different geometries (the coefficients describing the accumulation of radiation in the first layers and attenuation ofradiation with an increase in the shielding thickness). In the1960s70s, attheJINR Synchrocyclotron and Synchrophasotron, M. M. Komochkov, V. N. Lebedev, and V. E. Aleinikov performed a cycle of studies of radiation fields beyond the shielding and intheenvironment of the accelerators. It was found already in the beginning oftheresearch that neutrons are the most penetrating component of secondary radiation. It is neutrons of a wide energy spectrum that determine theradiation dose received by experimenters and other staff beyond the shielding during the operation of accelerators.

To determine the mechanism of scattered neutron field formation beyond shielding, experiments were performed at the JINR Synchrocyclotron and Synchrophasotron to study secondary high-energy neutrons (that are generated inphysical targets by proton beams) passing through local shieldings made of different materials. A particular difficulty consisted in the necessity to develop specific techniques of field measurements for studying the characteristics of scattered radiation fields beyond shieldings. In particular, thebroadest energy range multisphere neutron spectrometer, liquid and plastic scintillator-based high-energy neutron radiometers, a neutron dosimeter (remmeter), a recombination ionization chamber (designed by M. Zelchinsky), and a calibration ruler for metrological support of measurements were designed and produced. Also, the methodology of predicting accelerator radiation environment was developing. For example, B. S. Sychev worked out a method of calculating neutron radiation shieldings based on solving a system of integro-differential (kinetic) equations of radiation transport in matter; M. M. Komochkov, V. N. Lebedev, and L. N. Zaytsev developed an engineering (semiempirical) technique of estimating neutron fluence and dose values beyond shieldings.

Another rather difficult problem was that a portable personal dosimeter had to be created which would correctly measure personal doses in a multicomponent (neutrons, gamma rays, and charged particles) scattered radiation field withabroad energy spectrum. The Soviet-made personal dosimeters were nonserviceable inaccelerator radiation fields. Much credit goes to M. I. Salatskaya (thenHead ofthePersonal Dosimetry Monitoring Group at the RPD) for the development of a combined gamma and neutron dosimeter on the basis of the MK-20 emulsion and an X-ray film (the IFKn cassette). In a number of international collations, theadequacy ofits equivalent dose readings was confirmed. Possibilities were also studied of using other types of detectors to measure personal doses in particular, LiF-based thermoluminescent detectors.

The experience of radiation monitoring of accelerators acquired by JINRs RPD was unique to the Soviet Union; therefore, the basis of the RPD at the U-70 accelerator, the Institute of High Energy Physics (IHEP), was formed by JINR's RPD specialists who moved from Dubna to Protvino. Due to this, along with the community ofthe problems being solved, the cooperation and contacts between the two similar subdivisions of JINR and IHEP have been closest and most fruitful.

In the 1970s80s, further radiation research was mainly aimed at the accumulation of experimental data and, in parallel, development of techniques of calculating radiation transport through shielding. It was assumed that protection physics would develop in close connection between experimental and theoretical research, which supported confidence in the reliability of predicting the situations at the facilities that were then being designed with continuously increasing beam powers and accelerated particle energies. But the great amount of experimental data that had been acquired by that time could not have been used to test the adequacy of the calculation techniques and were in fact empirical. It became clear that benchmark experiments in protection physics had to be performed in simple (idealized) geometries which, at the same time, would be typical for accelerators and correspond to all the initial information necessary for adequate calculations. Of principal importance was the detailed knowledge of the source terms, especially for heavy ion accelerators, because there had been no data on the production ofsecondary neutrons in nucleusnucleus interactions. Such experiments were performed beyond the LNP Synchrocyclotron shielding and at the relativistic particle beams of the Synchrophasotron, the Laboratory of High Energies (LHE).

It was inexperiments atthe Synchrocyclotron (G. N. Timoshenko) that for the first time double differential by angle and energy yields of charged particles from shielding were obtained, and the charged component contribution to the total dose and fluence was evaluated. For these purposes, a compact dE/dx charged particle spectrometer was made; its calibration was performed based on elastic p- p scattering atabeam oftheLNP Phasotron. Using a system of sensors for measuring the angular distributions ofcharged particles, regularities were studied of the formation of radiationfields indifferent geometries beyond the Phasotron and Synchrophasotron shieldings.

In comparative experiments at proton, alpha particle, and 3.65-GeV/nucleon 12C beams, initial data were obtained on the yield of secondary charged particles from thick Cu and Pb targets. Using the time-of-flight method, spectra of secondary neutrons with energies above 10MeV were measured at different angles intheinteraction of relativistic nuclei with a thick target for the first time. These results were used to check the calculations of particle transport in matter and to predict the radiation environment at the Collective Heavy Ion Accelerator and Nuclotron as part of their design. To calculate the shieldings for accelerators of nuclei, A. R. Krylov developed software which modeled an internuclear cascade in thick targets based onthe firestreak model of nucleusnucleus interactions and a program forcalculating neutron transport in shielding based on solving a system of kinetic equations.

Thereliability of calculating the primary and secondary radiation fields was evaluated in a series of experiments in particular, in a benchmark experiment inprotection physics performed by G. N. Timoshenko beyond a relatively thin catcher ofa3.65-GeV/nucleon beam of 12C nuclei at the LHE Synchrophasotron.

A notable contribution to the methodology of physical measurements was made by V. P. Bamblevsky, who had mastered activation techniques and created a set oflow-background gamma-spectrometers for detector activity measurement.

Special attention was given to the development of neutron spectrometry inabroad energy range as the basic method of studying the radiation environment and measuring the neutron dose rate. The reconstruction of neutron spectra based on themultisphere spectrometer readings belongs to the class of inverse problems.

Itconsists infinding the unknown quantity on the grounds of a number of known effects and isreduced to solving a system of algebraic equations. Inthebeginning of neutron spectrometry research, for the uniqueness of the solution the spectrum was represented apriori by a linear combination of several known functions (the Maxwell distribution of thermal neutrons, the 1/E falling of the moderated neutron spectrum, evaporation spectra of different temperatures, etc.); that is, information onthecharacter of the solution was a priori very rigid. Later, thestatistical regularization method, developed in the 1970s by Acad. A. N. Tikhonov, began tobeused to reconstruct the spectra. This method requires minimal information apriori. Aprogram was developed of neutron spectrum reconstruction from readings of different modifications of the multisphere spectrometer (with an active thermal neutron detector and activation detectors). The neutron spectrum reconstruction technique was then being improved to extend the spectrometers operating range to high energies of neutrons (hundreds of MeV), as well as to increase the precision of calculating the sensitivity functions and check them experimentally.

Thus, in the early 1980s, at the IBR-30 reactor beams and at the neutron generator, experimental measurements were performed of thesensitivity functions ofthemultisphere spectrometer and other neutron detectors used in on-line radiation monitoring. Nevertheless, the multi-sphere spectrometer, due to its specifics, yields low-value information onhigh-energy neutrons, which largely limited its applicability in the measurements in high-energy radiation fields beyond the Phasotron and Synchrophasotron shieldings. For solving this problem, G. N. Timoshenko and A. R. Krylov proposed an original highly sensitive technique of high-energy neutron spectrometry in scattered radiation fields. Based on this technique, a neutron spectrometer of a new type was developed, had its sensitivity functions calculated, and was calibrated. With its help, a great amount of hard neutron spectrum measurements were done beyond JINRs shieldings and the readings of the stationary neutron radiation monitors immediately at work places were corrected. The high neutron sensitivity of this spectrometer also allowed aspectrum of space neutrons with energies above 20MeV to be measured on the Earths surface. In recent years, thedevelopment of neutron spectroscopy consists in theimprovement of calculating the sensitivity of the multisphere spectrometers on thebasis ofmodern Monte Carlo particle transport software MCNP, inclusion of heterogeneous spheres in thespectrometer configuration, and acquiring the experience of neutron spectrum reconstruction based on readings of activation detectors. The multisphere spectrometer was also used for the first time for studying secondary neutron fields around a thick target irradiated with 660-MeV protons. A 8cm thick and 50cm long lead target was imitating the core of a subcritical assembly monitored by a proton beam of the LNP Phasotron (the SAD project). This methodology of spectrometry allowed obtaining spectral and angular distributions of neutrons from the target in the whole energy range beginning with tens of keV. This experiment was performed to check internuclear cascade calculations using the most common transport software.

As was already noted, the multisphere spectrometer was in fact not only themain instrument for studying scattered radiation fields, but also a reference instrument of radiation monitoring. But metrological support to accelerator radiation research needs both a reference measurement instrument and a reference source ofneutron radiation. In practice, it is 239PuBe and 252Cf radioisotope sources with theaverage neutron energy of 4.3 and 2.5MeV, respectively, that areused asreference neutron sources. As regards metrology, their main disadvantage is theirnarrow energy range not matching the real radiation fields beyond shieldings, which does not provide thenecessary precision of practical measurements. In the 1980s, it led tothedevelopment of special metrological support of neutron measurements that was based onthegeneration of reference neutron fields of a wide energy spectrum immediately at the nuclear physics facilities and reproduction of the special State standard units there (by a direct or indirect method). The first reference neutron fields had been generated at reactors several years before and were used as reference neutron energy spectra. Onthe initiative of the subsection Radiation Protection and Work under High Levels of Ionizing Radiation of the Council onAccelerating Charged Particles of the USSR Academy of Sciences, which was then headed by M. M. Komochkov, a task was set to produce reference neutron fields at accelerators. This work was started in parallel at JINR and IHEP in the early 1990s and, later, at CERN. At JINR, four reference fields were generated: two, on the basis of252Cf neutron sources surrounded by moderators of different diameters; and two, on the basis of real fields of the LNP Phasotron. A soft reference neutron field was generated in the tunnel labyrinth on the basement floor under the main accelerator hall; a hard one, beyond the two-meter thick concrete shielding of the Phasotron atthebanking ofthewestern wall. The characteristics of these fields were measured in detail; systems of tracking their parameters were created; a methodological outline of calibrating dosimetric and radiometric instruments was proposed. In JINRs reference neutron fields, a collation was made of the neutron fields dosimetric and physical characteristic measurement techniques and the equipment used at JINR and the Institute of Atomic Energy (Swierk, Poland); also, a number of instruments and methods ofoperational and personal monitoring were calibrated.

A toughening of radiation standards and an increase in the amount of radiation monitoring at JINRs nuclear facilities required in the mid-1980s that the RPD took a new approach to the organization of zone monitoring namely, that automated radiation monitoring systems (ARMSs) be provided for JINRs facilities.

Itshould be noted that there was no experience in developing such systems for accelerators atthat time. The ARMS of the IBR-2 reactor had been developed similarly tothoseof the nuclear power plants. But the specifics of the radiation fields beyond accelerator shieldings, variability of accelerator operation modes, changes in thestatuses of the radiation monitoring zones depending on the operation modes, etc.

made itimpossible to use the nuclear power plant ARMSs at accelerators. To solve thistask, a three-level structure of an automated system was proposed: the first level included tens of stationary neutron and gamma sensors; the second, intellectual crate controllers to acquire information from the sensors and to control them; thethird, personal computers for data visualization and documentation and for controlling the system as a whole. The systems neutron channels with stationary wide-energy range neutron sensors based on corona counters in moderators, which had shown themselves to the best advantage during many years operation, were developed; special electronics units were created for the systems second level; software was developed for the systems second and third levels; a metrological scheme of sensor check and calibration was worked out. ARMSs with specific differences were provided forthe LNP Phasotron, LHE Synchrophasotron, and accelerators of the Laboratory of Nuclear Reactions, where, undergoing continuous improvement, they have been in operation for about 20 years.

Approximately in the mid-1980s, in parallel with ARMS development, work was begun to restructure the personal dosimetry monitoring (PDM) system. The traditional photometric monitoring methods based on using X-ray films to evaluate thegamma dose and nuclear emulsions to evaluate the neutron dose miss the necessary immediacy with the radiation-monitored JINR staff being about 2000.

Also, film and emulsion supplies became irregular. It was suggested that the PDM methodology be radically changed and monitoring be done, in part or in full, withthermoluminescent detectors (TLDs). As an alternative to the film dosimeter, the albedo neutron dosimeter with two 6Li- and 7Li-based TLDs was proposed, which detects soft neutrons rescattered from the body into the dosimeter. The RPD began the development of such a dosimeter and the automation of processing its data. Inthe1970s, when there were no industrial instruments for reading TLD data, theRPD tried todesign its own instrument of this kind. During the development ofan albedo dosimeter, different types of TLDs were tested; the dosimeter sensitivity calculations and calibration were performed; the dosimeter was tested in real neutron fields. Duetothedisadvantages inherent in the TLDs, it turned out to be impossible tocompletely drop photometric monitoring. Thus, a new combined PDM cassette was designed (itsTLD is processed using equipment produced by the Harshaw company).

The Departments experience in accelerator shielding development and methodology of calculating particle transport in matter were used to design the shielding of several facilities and buildings of JINRs accelerators. The RPD participated in the design of different versions of LHEs heavy ion accelerating complexes (the Collective Heavy Ion Accelerator and Heavy Ion Accelerator Complex) and theNuclotron. Inthe late 1980s, the Departments specialists contributed to the development of Vina Institutes cyclotron (Belgrade, Yugoslavia).

After the establishment of the Department of Radiation and Radiobiological Research (1995), investigations on the dosimetry of different types of radiation and protection physics were conducted within the radiation research project. Its main fields included studying the performances of advanced detectors and dosimeters;

wide energy range neutron spectrometry; optimization of radiation safety measures and shieldings; physical support of radiobiological experiments; staff and environment radiation monitoring; and training specialists in radiation protection.

Extensive research was done concerning the performances of solid state track detectors and thermal neutron detectors in polyethylene moderators. In particular, in joint work with specialists of the Institute of Nuclear Physics (Prague, the Czech Republic), the efficiency of detecting heavy nuclei (C, Mg, Ar, and Fe) byaCR-39 track detector was measured.

In connection with the Slovak Cyclotron Center (SCC) project for accelerating ions with A 130 up to 72MeV/nucleon, it was necessary to do research and development to minimize accelerator-generated radiation influence on the environment. The shielding design and radiation protection measures had to meet rigid requirements due to the complex being located in the city of Bratislava. A radiation concept was developed for the SCC, which took into account the possible ionizing radiation sources, protection from different types of radiation, radiation monitoring, the management of radiation sources, the analysis of possible radiation disasters, and the SCC influence on the environment. Based on this concept, the radiation protection part of the SCC project was developed (V. E. Aleinikov, V. A. Arkhipov, G. N. Timoshenko, A. R. Krylov, L. G. Beskrovnaya).

The DRRR did much work to create devices for the precision dosimetry ofcharged particle beams of JINRs accelerators. For these measurements, an experimental channel and an automated sample change facility were made at the U-200 accelerator.

Also, a methodology of measuring the absorbed low-energy ion dose wasdeveloped.

For experiments at the LHE Nuclotron, a methodology was developed oftheformation of a quasiplanar dose field and the measurement of the absorbed dose insamples, which allowed a cycle of research to be done atthe proton, alpha particle, and carbon and magnesium nuclear beams with energies of 0.51GeV/nucleon.

Besides carrying out the Departments own research, DRRR specialists actively worked in JINRs other fields: transmutation of radioactive waste produced bynuclear power plants; designing stationary facilities for the detection and identification of explosives and drugs; etc.

LABORATORY OF RADIATION BIOLOGY

In 2005, by a resolution of the JINR Directorate, Scientific Council, and the Committee of Plenipotentiaries of the Governments of the JINR Member States, the Department of Radiation and Radiobiological Research (DRRR) was reorganized into the Institutes new the eighth Laboratory: the Laboratory of Radiation Biology (LRB; JINR Order No. 403 of 20 June 2005). This event was consistent with the long development of one of the fields of fundamental biology atJINR and came as recognition of the great contribution of the DRRR specialists to solving important scientific problems. It is clear that as an interdisciplinary science radiobiology needs support from physicists; in this regard, JINR offers unique opportunities because it has highly competent physicists, necessary equipment, and the widest spectrum ofvery diverse sources of radiation. In fact, no other scientific center, either inRussia or abroad, is better suited and equipped physically for conducting radiobiological research than JINR. The LRB has thus all the rights to claim tobe theleader in research on the genetic effects of ionizing radiations with different physical characteristics among other scientific institutions of Russia and JINR Member States. Prof.Evgeny Krasavin, Dr.Biol., was appointed in 2005 Organizing Director, and elected in 2009 Director of the new Laboratory by the JINR Scientific Council.

In 2008, at the proposal by Acad. A. I. Grigoryev, the Academician Secretary ofthe Section of Biological Sciences of the Russian Academy of Sciences (SBS RAS), E. A. Krasavin presented the results of radiobiological research conducted at JINR to a session of the SBS RAS Bureau, where they were highly appreciated. A general SBS meeting unanimously supported a proposal of the SBS RAS Bureau and resolved to provide the scientific and methodological supervision of LRB by SBS RAS (Resolution No. 5 of 15 December 2008). This step raised JINRs biological research to a new level, providing the use of the Institutes unique potential.

The LRB was formed by two Departments (Radiobiology and Radiation Research) and three separate Sectors (Photoradiobiology, Space Radiobiology, and Computer

Molecular Modeling). The Department of Radiobiology consists of four Groups:

Molecular Radiobiology, Radiobiology of Normal and Tumor Cells, Radiation Genetics, and Mathematical Modeling. The Department of Radiation Research includes two Groups: Research on the Radiation Fields of JINR Basic Facilities and Calculation of Physical Protection from Radiation Effects. The biological effects ofradiations with different physical characteristics remained the LRBs main field ofresearch. Its topicality, as said earlier, is determined by a number ofcircumstances first of all, the efficiency of using radiations in a wide range of linear energy transfer (LET) for solving different fundamental and practical problems. These problems are related to fundamental issues of radiobiology, radiation therapy with accelerated ions, improvement of the protection of staff working in mixed ionizing radiation fields, and protection of the spacecraft crews in the conditions of long interplanetary flights.

After the establishment of the LRB, comparative research on the regularities and mechanisms of the induction of gene and structural mutations by radiations inawide LET range was continued in experiments on prokaryotic cells with different genotypes (A. V. Boreyko). In early works on the mutation frequency in microorganisms under irradiation in the presence of physical, chemical, and biological factors, an important role of postirradiation recovery in the induced mutation process was shown.

After mutants with different DNA repair defects had been isolated and identified, itbecame possible to study closely the role of the repair processes ininduced mutagenesis. The obtained results allowed concluding that both physical and biological factors play an important role in the mutagenic effect of radiations. The role ofthe physical factor in the mutation process could be studied withtheuseofionizing radiations of different physical characteristics; thatofthebiological one, withtheuse of different repair mutants. In experiments on bacteria with different genotypes itwas found that nonlinear dose curves of mutagenesis areobserved quite often and are revealed in taking into account both reverse and direct mutations. Such dependences had long been a vexing problem for specialists. For this reason, experiments were planned at the LRB to study the mutagenic effect of radiations inawide LET range generated by JINRs accelerators on prokaryotic cells with different genotypes.

For understanding the mechanisms of the induced mutation process, an extremely important task is the comparative study of the regularities and mechanisms of the formation of both gene and structural mutations in cells under radiations inawide LET range. Indeed, to solve a number of topical practical and scientific problems related to the genetic effect of ionizing radiations of different physical characteristics, not only is it necessary to have information on the total yield ofmutations of different types in irradiated cells; the data on the frequency of both gene and structural mutation formation in cells with different genotypes are exceptionally interesting. Obtaining this kind of data is extremely difficult in experiments onmammalian cells. Of course, the acquisition of data on the yield of different types ofmutations induced by radiations of different qualities in experiments onmammalian cells is an imperative practical and scientific problem; though, research ofthis kind ondifferent types of bacterial cells is a necessary step in solving it. The structural and functional organization of bacteria had already been studied in detail; theirdifferent repair-deficient mutants had been obtained. This all allows ascertaining themolecular mechanisms of the formation of gene and structural mutations in cells under radiations in a wide LET range.

The obtained information on the mutagenic effect of radiations in a wideLET range allowed understanding the mechanisms underlying the revealed regularities.

On the basis of studying the induction of both direct and reverse gene mutations, itwas found that the dose dependences of the mutation frequency are linear-quadratic for different radiations. For irradiation doses above 80100Gy, a power dose dependence is observed. On a logarithmic scale, the dose dependences are straight lines with a slope ratio of1.71.8, which points to a near-quadratic power character of these curves. The most efficient mutation induction was observed in experiments with accelerated helium ions with a LET of 20keV/m; ions with greaterLET have lower mutagenic efficiency. The heavy charged particle dose dependence ofthemutation frequency maintaining a quadratic law is determined by a number of factors. A microdosimetric analysis of the revealed regularities (S. Kozubek) shows that three types of cell subpopulation can be singled out for different doses of ionizing radiations in an irradiated population: undamaged surviving cells; lethally damaged nonsurviving cells; and moderately damaged cells that complete the repair process and join the surviving cell pool. With increasing LET, the nondamaged cell fraction increases, and the fraction of lethally damaged cells hit by particle track cores decreases. Consequently, mutations mainly develop in the subpopulation damaged by delta-electron passage and in the small fraction of cells through which at least one particle passed hitting the cell with the track core, provided that these cells have repaired the induced damage and survived. On this basis, it became possible to explain the conservation of the character of the mutagenesis dose dependences forradiations of different LET. As the electromagnetic and corpuscular radiations donot differ in the character of delta-electron energy transfer to matter, the type ofthe Nm /N (D) dependence, where Nm /N is the ratio of the mutant cell number tothe total cell number in the irradiated cell population and D is the dose, remains the same for radiations of different qualities. Therefore, under exposure tohigh-LET heavy charged particles (LET 100 keV/m), when particle tracks pass through thecell sensitive structures, cells mainly die; in surviving cells, the so-called deltaelectron mutagenesis takes place. When cells are irradiated with accelerated light ions or high-energy charged particles of LET 100 keV/m, the sensitive structures areexposed to the direct effect of tracks; then mutagenesis of the track core type takes place. The above having been taken into account, it becomes clear why thecharacter of the dose curves of mutagenesis in Escherichia coli and Bacillus subtilis cells remains the same with increasing particle LET.

The data obtained at heavy ion accelerators allowed concluding that the quadratic character of the mutagenesis curves is explained by the necessity of the realization and interaction of two mutually independent hitting events. The first ofthemisconnected with the appearance of a premutation lesion in the locus under study; the second one is the formation of a lesion that induces the SOS repair system thesystem that facilitates the fixation of changes in the bacterial DNA chain as mutations. Since SOS repair is the deciding factor in the realization oftheinduced mutation process, the analysis of the molecular mechanisms of SOSsystem organization inE.coli bacteria is important. Based on this, a molecular model ofinduced mutagenesis was developed, which allows describing the main ways of the transformation of the primary disorders in the DNA structure (premutation lesions) intomutations (A. V. Boreyko). In this model, the fixation of an ionizing radiationinduced premutation lesion as a point mutation results from the functioning of different enzymatic mechanisms, one of the most important of which isa multienzyme complex that includes inducible DNA polymerase V (UmuD' C), RecA protease, SSBproteins, and DNA polymerase III subunits.

Based on the molecular model, a mathematical model of the ultraviolet radiation-induced mutation process in E. coli bacterial cells was created (O. V. Belov, E. A. Krasavin, A. Yu. Parkhomenko). Actually, a new approach to the theoretical description of induced mutagenesis in bacterial cells was proposed. It was thefirst time that a model of the induced mutation process was developed based on thedetailed mathematical description of the key protein interactions in the course oftheSOS response of E.coli bacteria. Within one model approach, the whole way was traced from the appearance of an original DNA structure lesion to its fixation asamutation.

The developed model concepts for the first time allowed prediction ofthedynamics of the concentrations of the umuD gene dimerized products and two regulatory complexes of the SOS system: UmuD2C and UmuDD'C. Such an approach allowed a detailed modeling of the translesion synthesis mechanism, which is responsible fortheprocess of fixing premutation lesions as mutations. Calculations based onthis model resulted in the detection of a link between the efficiency oftranslesion synthesis realization and gene mutation yield. Calculations performed ontheexample of the lacI regulatory gene in E. coli bacteria showed concurrence of the modeling results and experimental data on the UV radiation energy fluence dependence ofthelacI mutation frequency. On the grounds of these approaches, it is possible todo further mathematical analysis of the main types of the mutation process induced by ionizing radiations with different physical characteristics in E.coli cells.

Ofcourse, itisamuch more complicated problem, but it is quite possible to solveit.

The successful solution of this problem requires, first of all, experimental data onthekinetics of the production and degradation of the main gene products participating intheformation of a multienzyme complex: DNA polymerase V.

In later papers on research in quantitative radiobiology, approaches were developed that allow identification of additional mechanisms of bacterial cell repair after radiation exposure. In particular, a detailed mathematical description was proposed of the excision repair of damaged DNA bases in bacterial cells; and the mechanism was modeled of damage removal involving formamidopyrimidine glycosylase (theFpg protein), which has several activity types. In this research, thus, not only were significant results obtained on the quantitative estimation of the production and degradation kinetics of the main gene products in the course of DNA repair, buta new theoretical approach to the description of the induced mutation process inbacterial cells was successfully realized.

In experiments with accelerated heavy ions, it was shown that the frequency of the deletion mutations, unlike that of the gene mutations, increases linearly with the dose for all the used types of radiation. The most effective are ions ofLET 50keV/m. Accelerated heavy ions of higher LET have a weaker biological effect. Therefore, the character of the dose dependences by the criterion of deletion mutation induction in E.coli cells is completely different from the character ofthedependences obtained for gene mutations, which were examined earlier. Inthelatter case, a near-quadratic power dependence is observed. The dose dependences of deletion mutation induction, which are described by linear functions, aredetermined by other mutation formation mechanisms than the dose dependences of gene mutation induction. The linear character of the dose dependence of deletion formation inbacterial cells under gamma irradiation is explained by the fact that it is DNA double-strand breaks (DSBs) that is the molecular basis of the primary lesions leading todeletions (unlike that, it is base damage that is the molecular basis ofthe primary lesions leading to gene mutations). To turn premutation lesions of the DSB type into a structural mutation, there is no need in the induction of the SOS repair system, which, as shown earlier, plays the key role in gene mutation formation.

On the basis of the performed research, it was shown that the biological effectiveness of heavy charged particles evaluated by the induction of deletion mutations increases with increasing LET, like it does if evaluated by the lethal effects of irradiation and point mutation induction. But the location of the maximums oftheLET dependence of the relative biological effectiveness (RBE) of the considered irradiation effects isnot invariant. With respect to the lethal effect, the highest RBE levels are observed for irradiation with particles of LET 100keV/m. By the criterion of gene mutation induction, the maximum is located at 20 keV/m. For deletion mutations, this value is LET 50 keV/m. On these grounds, a conclusion was made that thedifferences in the maximum locations of the RBE(LET) dependences forthelethal and mutagenic effects of radiation are determined by the different character of the DNA lesions participating in the realization of gene mutagenesis and lethal effects. Intheformer case, those are mainly damaged bases; in the latter, DNADSBs. The microdosimetric analysis of the LET dependence of clustered single- and double-strand break yield shows that both types of dependences are described by curves with a local maximum (V. Michalik). The clustered single-strand break RBE maximum, however, is shifted by almost an order of magnitude to lower LET, which can explain the difference in the locations of the maximums of the RBE dependences onLET for the lethal effects of radiation and gene mutation induction.

Experiments on the induction of mobile elements by radiations of different physical characteristics (A. V. Boreyko, D. V. Zhuravel) were planned taking into account the fact that the precise excision of transposons, while being a deletion process byitsmolecular nature, depends on the functions of the genes controlling SOS repair. As the formation of the deletion mutations, which are based on DNA DSBs, isnotdetermined by inducible SOS repair, it seemed important to study the regularities and mechanisms of the precise excision of transposons in E.coli bacteria induced by gamma radiation and accelerated heavy ions of different physical characteristics.

With increasing the dose of irradiation with heavy charged particles, thefrequency of the deletions caused by the precise excision of the mobile element was described by power dependences. With increasing LET of the particles, their biological effectiveness compared to that of gamma rays increased, and theRBE maximum calculated by the criterion of the precise excision of the Tn10 transposon was realized intheLET range of 2040keV/m. As was mentioned before, themaximal gene mutation yield in E.coli and Bacillus subtilis bacteria is observed atthesame LETvalues.

Onthegrounds of the obtained data, a conclusion was made that thehigh biological effectiveness of heavy charged particles with respect to the induction of mobile elements and gene mutations is determined by two circumstances. The precise excision of the transposon is on the one part a deletion event; on the other, itisdetermined bySOS-dependent mechanisms. The transposon excision initiation isbased ontheformation of single-strand pins in its sequence, which are produced in the course of DNA damage repair; and the formation of a lesion that triggers cell SOS response, which leads to the excision of the mobile element. Thedifference inthe characters of the premutation lesions the molecular substrate for the formation of the gene and structural mutations has an effect onthe character oftheRBE dependences on LET. Clustered lesions of a DNA strand that appear against the cell SOS response background can be the molecular basis oftrasposon excision. The evidences of this circumstance are the power character ofthe dose dependence of the induction of mobile elements by radiations of different physical characteristics and the position of a local RBE(LET) dependence maximum determined by this criterion, which correlates with the similar dependence forthegene mutations.

By the establishment of the LRB, the Molecular Radiobiology Group had started research on molecular disorders in the human lymphocyte DNA structure under irradiation with gamma rays and accelerated heavy ions and research on apoptotic cell death. With the use of the DNA comet assay, regularities were studied in the induction and repair of DNA DSBs in cells irradiated with 60Co gamma rays and accelerated 7Li and 11B ions with LET of 20 and 40keV/m, respectively (A. V. Boreyko, V. N. Chausov, V. A. Tronov). For gamma rays and accelerated ions, the dose dependences were found to be linear; it was shown that heavy ions have greater biological effectiveness than gamma rays by the criterion of DNA DSB induction. The RBE of accelerated Li and B ions is 1.4 0.1 and 1.6 0.1, respectively. To study the qualitative specifics of DNA DSBs induced in cells by radiations in a wide LET range, anapproach involving the use of agents that influence DNA synthesis was used atthe LRB. It is known that a number of DNA synthesis inhibitors (cytosine arabinoside, oxyurea, fluorodeoxyuridine, and some others) suppress not only replicative, but also reparative DNA synthesis in mammalian cells.

In the presence ofsuch agents, a significant increase in cell sensitivity to gamma radiation isobserved inthepostirradiation period. The molecular mechanism oftheir sensitizing effect consists in blocking the build-up of single-strand gaps in the DNA chain. Asa result, the opposite DNA strand with continuously unrepaired gaps can be attacked by S1 endonucleases, and enzymatic DSBs will form. Along with this, there isnoinfluence oftheDNA synthesis inhibitors on mammalian cell survival under radiations with high LET. This prompted a suggestion that with increasing particle LET, theyield of direct DNA DSBs induced immediately by heavy charged particles significantly increases, and their yield is determined only by the physical properties ofradiations. In this connection, it seemed important to study the regularities intheeffect of DNA synthesis inhibitors on the yield of DSBs induced incells by radiations in a wide LET range. The data obtained on the effect of DNA synthesis inhibitors (cytosine arabinoside (Ara-C) and hydroxyurea (HU)) on DNA damage induction and repair indicated that the character of their modifying effect is different undercell exposure toionizing radiations of different quality. It was observed that under normal conditions DNA DSBs are induced more efficiently by heavy ions, theRBE of which is 1.6 0.1. The obtained results also pointed to the efficient repair of DNA DSBs induced by the used radiations. In the presence of inhibitors, significant differences were observed in the character of the established doseeffect dependences for cell irradiation with gamma rays and accelerated boron ions. For gamma irradiation in the presence of DNA synthesis inhibitors, not only DNA DSB repair was not observed, but DSB yield increased somewhat with cell incubation time. It could be explained, on the one part, by possible inhibition of DNA DSB repair, which, as is known, is realized by two mechanisms: homologous recombination(HR) and nonhomologous end joining (NHEJ). Ontheother part, it could beconnected with theformation of enzymatic DNA DSBs from DNA single-strand breaks (SSBs), which emerge during the incision of modified nucleotides in the process of excision repair. As the removal of damaged nucleotides in mammalian cells in the process of excision repair lasts 34 hours after irradiation, the SSBs forming in the presence of DNA synthesis inhibitors can become the sites for enzymatic DSB formation caused byS1 endonucleases attacking DNA strands that are opposite toincision SSBs. When cells are irradiated with accelerated boron ions in the presence ofAra-C and HU, DNA DSB repair is observed, which is not so in the case ofgamma irradiation; thelower values of dose change factor parameters in the case of cell irradiation with heavy ions are explained by a decrease inthenumber oftheinduced DNA SSBs with increasing LET of radiations. It is exactly this type of DNA damage that makes up the molecular substrate for the realization of the sensitizing effect ofthe used DNA synthesis inhibitors.

Thus, on the grounds of the conducted research, it was shown that when mammalian cells are exposed to ionizing radiations in the presence of DNA synthesis inhibitors Ara-C and HU, DNA DSB repair takes place, which was observed forboth gamma rays and accelerated boron ions. The great contribution of enzymatic DNA DSBs developing from inhibitor-blocked joining groups of the direct SSBs and enzymatic SSBs forming in the course of excision repair seem to overlap DSB repair under gamma radiation, which is observed when cells are irradiated with accelerated boron ions. On these grounds, experiments were planned at the LRB with heavier charged particles of yet higher LET ( 200 keV/m) which induce mainly direct DNA DSBs, while the enzymatic DSB contribution is minimal.

For studying regularities in the formation of DNA damage of different types under ionizing radiations of different quality, the enzymatic DNA comet assay technique was developed. The use of the enzymes of endonuclease III (EndoIII) and formamidopyrimidine glycosylase (Fpg) repair makes it possible to transform modified pyrimidine and purine bases into DNA SSBs. With the use of modifying enzymes in alkaline and neutral DNA comet assay, comparative dose dependences were obtained of the formation of DNA SSBs and modified purines and pyrimidines, as well as DNA DSBs and clustered DNA DSBs under 60Co gamma irradiation.

As was already noted on different occasions, accelerated heavy particles induce many effects that are strongly different from the ones induced by electromagnetic radiations. To a large extent, it is connected with the specifics of heavy charged particle energy transfer to the cell genetic structures. When cells are irradiated with gamma rays, the absorbed dose is delivered to the matters volume as numerous randomly distributed acts of energy transfer. The same dose of radiation can be transferred to the same matter's volume by a single heavy charged particle passing throughit.

This character of heavy ion energy transfer to the genetic structures determines the formation of DNA damage types that essentially differ from the ones typical fortheelectromagnetic ionizing radiations. Among the most severe heavy ion-induced lesions, considered first should be the DNA double-strand breaks (DSBs).

A heavy charged particle crossing a DNA section results not only in the violation oftheintegrity ofDNAs two complementary strands, but also in the damage ofother molecular structures adjacent to this site. For cell repair systems, such clustered lesions are the most difficult to repair. They are the molecular substrate ofcell death, induction of different types of chromosome mutations, and malignant transformations. The necessity of studying the regularities and mechanisms oftheformation and repair ofsuch a damage is clear. Therefore, the Molecular Radiobiology Group started research on the induction of DNA DSBs in human cells by radiations withdifferent physical characteristics and their repair. Efficient modern techniques were used

which allow studying the formation ofDNADSBs inthenuclei of individual cells:

immunocytochemical cell staining by protein-specific antibodies conjugated with different fluorescent dyes (the method ofDNAfoci) and the DNA comet method.



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